Entry and intracellular transport of hepatitis B viruses have several unusual, largely unknown aspects. In this study, we explored the mode of virus entry using the duck hepatitis B virus (DHBV) and the primary hepatocyte infection model. Upon internalization, viral particles were enriched in an endosomal compartment, as revealed by biochemical and ultrastructural analysis. Virus-containing vesicles harbored early endosome markers. Kinetic analysis revealed time-dependent partial translocation of viral DNA from endosomes into the cytosol. This was strongly reduced by inhibition of vacuolar ATPase; (vATPase) activity with bafilomycin A1 and resulted in abortive infection and prevention of cccDNA formation. Inactivation of vATPase induced accumulation and stabilization of incoming viral particles in endosomes, presumably by blocking endosomal carrier vesicle–mediated cargo transport and sorting. Although neutralization of the endomembrane organelles alone led to stabilization of incoming viral particles, it did not inhibit virus infection. In line with this, a pH-dependent ectopic virus fusion at the plasma membrane could not be artificially induced. This provided further evidence for a pH-neutral translocation mechanism. Endosomal membrane potential was required for viral infection because cotreatment of cells with monensin partially overcame the inhibitory effect of bafilomycin A1. In conclusion, hepatitis B viral infection is mediated by a novel cellular entry mechanism with features different from that of all other known viruses. (HEPATOLOGY 2006;44:685–693.)